Cyclic AMP has profound effects on flagellar motion and has been implicated as a key regulatory molecule in the initiation of mammalian sperm motility. The long term goals of this project are to characterize the molecular and biochemical mechanisms which localize protein kinase A (PKA) to specific flagellar structures, thereby targeting cAMP dependent phosphorylation reactions to specific regions in sperm. The flagellar PKA is associated with both the fibrous sheath and mitochondria via its regulatory subunit RII. Association of RII to the fibrous sheath is mediated through interactions with two sperm specific A Kinase Anchor Proteins (AKAPs). Two developmentally regulated testis specific AKAP cDNAs have been isolated. No other developmentally regulated AKAPs have been described. Experiments are being conducted to characterize the structural domains necessary for binding of RII to these sperm/testis specific AKAPs. The determinants on the AKAPs necessary for association with other fibrous sheath polypeptides will be determined. The hypothesis that the RII binding domain of each AKAP on the fibrous sheath directs PKA to phosphorylate distinct flagellar polypeptides will be tested. In addition to providing information on the structure and function of the sperm AKAPs, these studies also have more general significance, since AKAPs are important regulatory proteins in brain and other tissues. Since the association of RII with mitochondria is not mediated via an AKAP pathway, mass spectrometry will be used to identify and characterize post translational modifications on RII which may be responsible for association of PKA with this organelle.